Malaria continues to be one of the prevalent causes of morbidity and mortality around the world. Inhibitors of de novo pyrimidine metabolism may be suitable as antimalarial agents since the blood stage forms of Plasmodium lack enzymes to salvage preformed pyrimidine. In the past, the PI has demonstrated that a combination of 5-fluoroorate (5-FO) and uridine allowed for a 1000-fold difference in IC between malarial and mammalian cells in culture, and the PI showed that such a combination was curative in animal models of malaria. The nanomolar concentrations of 5-FO that caused inhibition of parasite proliferation also inactivated malarial thymidlate synthase (TS) activity and triggered cell death. In order to understand the potent cytocidal activity of 5-FO against malarial parasites, the present studies will identify the biochemical parameters that permit efficient activation of 5-FO to 5- fluorophyrimidine nucleotides in P. falciparum. To further understand the role of TS inactivation in the antimalarial activity of 5-FO, chemical modifications of TS will be characterized and 5-FO treated parasites will be examined for metabolic perturbations that are commonly associated with "thymineless death". The frequency of 5-FO resistance and the molecular basis for 5-FO resistance will be evaluated in P. falciparum. Finally, the antimalarial potency of folated-based TS inhibitors will be evaluated in the presence of thymidine. A careful study of TS inhibition in P. falciparum will not only lead to novel forms of malaria chemotherapy but will also shed light on the remarkable success of traditional antimalarial agents that interfere with two related steps: folate biosynthesis and reduction of dihydrofolate. This RO-1 application is a continuation of a FIRST award (R29) and will complement a Research Career Development Award (K04).